Control mechanisms are of crucial importance for the maintenance of normal metabolism and a knowledge of the molecular details of the mechanisms that regulate metabolism is essential for elucidation of pathological processes. Thus, knowledge of the molecular mechanisms for regulation of the urea cycle, the major pathway of ammonia removal, will provide an understanding of Reye's syndrome and other disorders (e.g., hepatitis, cirrhosis, and metabolic defects) in which liver function is temporarily or permanently altered. Carbamoyl phosphate synthetase, which catalyzes the entry step of the urea cycle, is known to be the primary site of control. However, the molecular mechanisms for this control have not been elucidated. Previous studies have established that carbamoyl phosphate synthetase is composed of independently- folded structural domains and have provided a map of these domains. It has also been established that a large portion of the enzyme is bound to the inner mitochondrial membrane. The goal of the proposed research is to develop a model for the overall carbamoyl phosphate synthetase structure and its role in function. The structural domains will be separated under nondenaturing conditions and it will be determined whether the individual domains possess full or partial enzymatic activity. The enzyme binding sites for the allosteric effecto N-acetylglutamate and for the ATP analogs 8-azido-ATP and p-fluorosulfo- nylbenzoyladenosine will be identified by proteolytic digestion of the labelled enzyme and amino acid sequencing of the fragments. The self-association of carbamoyl phosphate synthetase will be investigated and the regions of contact between monomeric units defined. The structural and functional aspects of the interaction of carbamoyl phosphate synthetase with the inner mitochondrial membrane will be further defined by in vitro binding and kinetic studies, as well as by in situ immunocytochemical studies.